Pigment epithelium-derived factor (PEDF) is a member of the serine protease inhibitor superfamily by sequence homology that has neurotrophic properties, but does not have a demonstrable inhibitory activity against serine proteases. Work in this research group is aimed at elucidating the mechanism of action of PEDF. The isolation and characterization of a PEDF receptor have been the focus of attention during the past year. The human retinoblastoma Y-79 cell line is used as a model system since PEDF induces neuronal differentiation in these cells. The saturation and Scatchard binding analysis of radiolabeled PEDF binding by Y-79 cells with increasing concentrations of unlabeled ligand revealed that PEDF exhibits a saturable and specific binding to a single class of receptors on the surface of retinoblastoma cells. A purification protocol was optimized for a PEDF-binding protein from membranes of Y-79 cells and of bovine and monkey retina. Determination of the amino acid sequence for a PEDF-binding protein is in progress. PEDF is a natural extracellular protein of the interphotoreceptor matrix (IPM), which contains glycosaminoglycans that confer a polyanionic nature to this matrix. PEDF is associated with the IPM by ionic interactions. We studied the nature of these interactions at a molecular level. Native and correctly folded PEDF binds to heparin, heparan sulfate and chondroitin sulfates A, B and C, while unfolded PEDF protein loses these binding affinities. Chemical modification and limited proteolysis were used to locate a glycosaminoglycan binding site on PEDF. In collaboration with Craig Hyde, NIAMS, the ionic surface potential was determined for a PEDF structural model that was developed from the known structures of serpins antithrombin III and ovalbumin. The folded protein conformation of the PEDF model has a cluster of exposed basic amino acids, lysines and arginines, available to interact with polyanions and glycosaminoglycans that constitute the glycosaminoglycan binding site of PEDF. This site is located opposite the neurotrophic active region and distant from the homologous serpin reactive loop. The preparation of crystals for the solution of the structure of PEDF by x-ray crystallography is in ongoing. We are collaborating with Lucien Houenou to study the effect of PEDF on motoneurons. PEDF rescues motoneurons from natural cell death and after axotomy. PEDF also promotes neurite-outgrowth in single cell cultures of embryonic avian motoneurons.